There are a number of patients who are on pancreatic supplements for unclear reasons or for whom the proper dose is unknown. The dose can range from three capsules per day to 24 capsules per day at a cost of approximately $0.30 per capsule. Due to actual or perceived difficulty with current testing methods, when physicians suspect pancreatic insufficiency, they will empirically place patients on different amounts of pancreatic enzyme and, if the patients fail to gain weight or their symptoms are not controlled, they arbitrarily increase the amount of enzyme, often without any confirmatory evidence that the patient's symptoms are due to pancreatic insufficiency. Abdominal pain, weight loss and diarrhea are very common symptoms of many different ailments that have nothing to do with small intestinal malabsorption or pancreatic dysfunction. One of the most often-used tests for pancreatic dysfunction is the bentiromide test. This test leads to misdiagnosis in at least twenty percent of cases. (Chymex package insert, Adria Laboratories, 1988, incorporated herein by reference, and data presented herein.) Because of its low sensitivity, the bentiromide test has been useful only for confirmation of severe pancreatic dysfunction (E. P. DiMagno (1982), "Diagnosis of Chronic Pancreatitis: Are Non-Invasive Tests of Exocrine Pancreatic function Sensitive and Specific?" (editorial), Gastroenterol. 83:143-146).
The standard bentiromide test for pancreatic insufficiency is a test which is often used because it does not require patient intubation or expensive endoscopy. Bentiromide (brand name Chymex) is a diagnostic agent marketed by Adria Laboratories, Columbus, Ohio. Its chemical formula is N-benzoyl-L-tyrosyl-p-aminobenzoic acid. This compound, together with analogous compounds also suitable for use in testing pancreatic functioning, is described in De Benneville U.S. Pat. No. 3,893,992 issued Jul. 8, 1975, incorporated herein by reference.
Bentiromide consists of a para-aminobenzoic acid (PABA) moiety attached to a synthetic peptide by a chymotrypsin-labile bond. Chymotrypsin is a pancreatic exocrine enzyme widely held to be the first enzyme to have decreased output with developing pancreatic insufficiency. Bentiromide is typically administered to a fasting patient in 500 mg. amounts. The patient is then encouraged to drink large amounts of water, and urine is collected over the following six hours. Measurement of excretion of the PABA portion of the bentiromide is then measured by a 1939 assay for arylamines modified in 1945. (Chymex package insert, Adria Laboratories, 1988).
The assay for arylamines is quite nonspecific, as there are numerous arylamines in urine and serum. If the test gives a low value suggesting pancreatic exocrine insufficiency, the possibility of problems in PABA absorption and excretion must be ruled out (C. Lang, et al., "Assessment of exocrine pancreatic function by oral administration of N-benzoyl-L-tyrosyl-p-aminobenzoic acid (Bentiromide): 5 years' clinical experience" (1981) Br. J. Surg. 68:771-775), and the patient is then required to return in about a week to repeat the procedure with PABA only (C. J. Mitchell, et al. (1979), "Improved Diagnostic Accuracy of a Modified Oral Pancreatic Function Test," Scand. J. Gastroenterol 14:737-741; Z. Weizman, et al. (1985), "Bentiromide Test for Assessing Pancreatic Dysfunction Using Analysis of Para-Aminobenzoic Acid in Plasma and Urine," Gastroenterology 89:596-604; V. S. Hubbard, et al. (1984), "Diagnostic and Therapeutic Applications of Bentiromide Screening Test for Exocrine Pancreatic Insufficiency in Patients with Cystic Fibrosis," Dig. Dis. and Sci. 29:881-889). The second procedure is used to correct for factors such as difficulties in absorption and excretion of PABA which can affect the amount of PABA present in the urine or serum.
The standard bentiromide test as applied to urine is inconvenient and often inaccurate because of the required six-hour urine collection, interference by common medications, and alterations in gastric emptying and renal function. Potential inaccuracies also occur because the assay is not specific for PABA. The above difficulties may account for the greater than twenty percent overlap between normals (range 50-90% recovery) and those with pancreatic disease (range 10-70% recovery); this is a significant problem.
The bentiromide test is useful to diagnose pancreatic insufficiency in children. A modified dose of 0.015 g/kg body weight has been used, and PABA recovery measured in faeces. (M. Sacher, et al. (1978), "PABA screening test for exocrine pancreatic function in infants and children," Arch. Dis. in Childhood 53:639-641). The bentiromide and xylose tests are described in attempts to evaluate pancreatic function and malabsorption in cats (E. C. Hawkins, et al. (1986), "Digestion of bentiromide and absorption of xylose in healthy cats and absorption of xylose in cats with infiltrative intestinal disease," Am. J. Vet. 47:567-569). Pancreatic insufficiency is correlated with cystic fibrosis. (S. Nousia-Arvanitakis, et al. (1978) "Diagnosis of exocrine pancreatic insufficiency in cystic fibrosis by the synthetic peptide N-benzoyl-L-tyrosyl-p-aminobenzoic acid," J. Pediatrics 92:734-737.) Some investigators have attempted to make the bentiromide test more sensitive by measuring PABA in serum rather than urine at different time points after ingestion of bentiromide. (S. A. Gharbo, et al. (1985), "Colorimetric Plasma Assay for the Bentiromide Test (BT-PABA) for Exocrine Pancreatic Insufficiency," Anal Biochem. 148:228-232; Z. Weizman, et al. (1985), "Bentiromide Test for Assessing Pancreatic Dysfunction Using Analysis of Para-Aminobenzoic Acid in Plasma and Urine," Gastroenterology 89:596-604; D. Laufer, et al. (1991), "The Bentiromide Test Using Plasma p-Aminobenzoic Acid for Diagnosing Pancreatic Insufficiency in Young Children," Gastroenterol. 101:207-213) Cystic fibrosis patients test abnormally low using the plasma bentiromide test. Measurement at 90 minutes post-administration is recommended to minimize error (G. Koren, et al. (1985), "Altered PABA Pharmacokinetics in Cystic Fibrosis," Dig. Dis. & Sci. 30:928-932; P. G. Lankisch, et al. (1986) "Pancreolauryl and NBT-PABA Tests, Are Serum Tests More Practicable Alternatives to Urine Tests in the Diagnosis of Exocrine Pancreatic Insufficiency?", Gastroenterol. 90:350-354).
The serum samples are generally analyzed for total arylamines by the Bratton-Marshall Reaction. (Bratton, A. C. and Marshall, K., "A new coupling component for sulfanilamide and other aromatic acids in dog and man," J. Biol. Chem. 128:537-550), although additional analytical tests have been proposed (H. T. Karnes, et al. (1984), "Determination of p-Aminobenzoic Acid in Urine by Room-Temperature Phosphorimetry, with Application to the Bentiromide Test for Pancreatic Function" Clin. Chem. 30:1565-1567; N. Bando, et al. (1990) "Enzymatic Method for Selective Determination of 4-Aminobenzoic Acid in Urine," Clin. Chem. 36:1937-1940; C. M. Riley, et al. (1985) "Analysis of N-Benzoyl-L-Tyrosyl-p-Aminobenzoic Acid (Bentiromide) Metabolites in Urine by Ion-Pair High-Performance Liquid Chromatography," J. Chromatography 338:377-388; C. M. Riley (1987) "Selected aspects of the development of methods for the analysis of drugs by high performance liquid chromatography," Xenobiotica 17:365-383; P. R. Durie, et al. (1992), "Bentiromide test using liquid chromatographic measurement of p-aminobenzoic acid and its metabolites for diagnosing pancreatic insufficiency in childhood," J. Pediatrics 121:413-416).
Another expedient for making the bentiromide test more sensitive is to stress the pancreas with additional protein (J. D. Berg, et al. (1986), "Exocrine Pancreatic Function as Determined in a Same-Day Test with Use of Bentiromide and p-Aminosalicylic Acid," Clin. Chem. 32:1010-1012; J. M. Braganza, et al. (1983), "Observations on the BT PABA/.sup.14 C-PABA tubeless test of pancreatic function," Clinica Chimica Acta, 130:339-347).
To solve the problem of having the patient return a week later for a separate PABA control test, it has been proposed to administer p-aminosalicylic acid with the bentiromide since its metabolism closely tracks that of PABA (J. D. Berg, et al. (1986), "Exocrine Pancreatic Function as Determined in a Same-Day Test with Use of Bentiromide and p-Aminosalicylic Acid," Clin. Chem. 32:1010-1012; J. W. L. Puntis, et al. (1988), "Simplified oral pancreatic function test," Arch. Dis. in Childhood 63:780-784). It has also been proposed to administer a radioactive isotope of PABA simultaneously with the bentiromide (J. M. Braganza, et al. (1983), "Observations on the BT PABA/.sup.14 C-PABA tubeless test of pancreatic function," Clinica Chimica Acta, 130:339-347); C. J. Mitchell, et al. (1981) "Preliminary evaluation of a single-day tubeless test of pancreatic function," Brit. Med. J. 282:1751-1753). Another test known to the art for measuring pancreatic insufficiency involves the use of radioactive isotopes of Vitamin B.sub.12 (U.S. Pat. No. 4,279,886 issued Jul. 21, 1981 to Robert H. Allen). However, these tests require administration of undesirable radioactive materials. Such radioactive materials cannot be administered in amounts stoichiometrically equivalent to the administered bentiromide, thus the sensitivity of the test is not as high as would be desirable.
Conversion of bentiromide to PABA by enzymes other than chymotrypsin does occur, but only in residual amounts (E. E. Sterchi, et al. (1988), "N-Benzoyl-L-tyrosyl-p-aminobenzoic Acid Hydrolase: A Metalloendopeptidase of the Human Intestinal Microvillus Membrane Which Degrades Biologically Active Peptides," Arch. Biochem. & Biophys. 265:105-118). Common intestinal bacteria also do not appear to convert bentiromide to PABA (K. Gyr, et al. (1978), "Chymotrypsinlike Activity of Some Intestinal Bacteria," Dig. Dis. 23:413-416). Thus, false negative results for pancreatic insufficiency with this test due to other sources of PABA derived from the administered bentiromide do not present a problem. It is, of course, necessary that other dietary sources of PABA be ruled out.
Patients with severe small bowel disease may have symptoms similar to those of patients with pancreatic insufficiency--diarrhea, steatorrhea or weight loss. Since PABA must be absorbed through the small intestine and partially conjugated by the liver, theoretically tests in patients with small bowel or liver disease could produce false positive results, i.e., decreased serum PABA after oral administration of bentiromide without pancreatic disease being present. The bentiromide test is generally believed to be reliably confirmatory of pancreatic insufficiency despite the possibility that abnormal results could occur in patients with small bowel disorders or liver disease (B. M. Meyer, et al. (1987), "Bentiromide Test Is Not Affected in Patients with Small Bowel Disease or Liver Disease," Pancreas 2:44-47), although the test does not show good specificity in gastrectomized patients (G. Heptner, et al. (1989), "Exocrine Pancreatic Function after Gastrectomy," Gastroenterol. 97:147-153). Liver disease does not interfere with the results until the disease is quite severe, the patient obviously jaundiced and not likely to be administered bentiromide. False positives due to small bowel disease may be readily detected using urinary absorption tests such as the xylose test or small bowel x-rays (P. P. Toskes (1984), "The Bentiromide Test for Pancreatic Exocrine Insufficiency," Pharmacother 4:74-80).
The serum xylose test is one of the best non-invasive methods for diagnosing intestinal malabsorption. In this test, D-xylose, a five-carbon sugar not naturally present in the body or in foodstuffs, is given orally and an estimation of the increase in serum xylose content is carried out through colorimetric determination (CD) of non-glucose reducing sugars in serum. However, the test is occasionally misleading by giving both false positive and false negative results. Older xylose tests measured xylose in the urine. Problems with this test include the fact that the Roe and Rice method of D-xylose analysis is non-specific and cross-reacts partially with glucose. Hence, this method is not accurate in the presence of hyperglycemia and glycosuria. Furthermore, the test critically depends on complete emptying of the urinary bladder at five hours and on intact renal function. False positive results are frequently obtained with both the serum and urine tests in patients with intestinal bacterial overgrowth (W. F. Caspary, "Diarrhoea and Carbohydrate Malabsorption," (1986) in Clinics in Gastroenterology, Vol. 15 Diarrhoea (G. J. Krejs, ed.) at 649-650 (W. B. Saunders Company, London, Phila., Toronto)), but delays in gastric emptying do not appear to affect the results (R. Stradley (1986), "Gastric absorption of D-xylose in the rat: its influence on the D-xylose absorption test," J. Lab. Clin. Med. 107:10-14).
An algorithm for evaluating further procedures using the bentiromide and xylose tests in patients with symptomatic diarrhea, steatorrhea or weight loss useful for diagnosing and assessing the necessity for further testing is presented in P. P. Toskes (1984), "The Bentiromide Test for Pancreatic Exocrine Insufficiency," Pharmacother. 4:74-80, incorporated herein by reference. Abnormal bentiromide with normal xylose indicates pancreatic exocrine insufficiency. If both tests are abnormal, primary small bowel disease is suggested, and a small bowel biopsy should be performed. Normal bentiromide with abnormal xylose indicates bacterial overgrowth and a .sup.14 C-xylose breath test and small bowel culture are indicated. If both tests are normal, the article states, pancreatic and small bowel disease are not ruled out, and further tests, such as the .sup.14 C-xylose breath test, small bowel culture, small bowel biopsy and secretin tests are indicated.
Stable isotopes have been used in metabolic investigations such as measurement of body water composition and energy expenditure and studies of substrate fuel metabolism in pregnancy, infants and adults (D. M. Bier (1987), "The use of stable isotopes in metabolic investigation", in Clinical Endocrinology and Metabolism, Vol I, No. 4 (Alberti et al., eds.) 817-836). Stable isotopes of PABA have not been previously known to the art.